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An international team has developed a method for
genetically altering induced pluripotent stem cells that could be useful for
treating inherited diseases.1 The team now plans to scale up the
method and will need to show the cells are safe for long-term use.

A
potential roadmap for using induced pluripotent stem (iPS) cells as
therapeutics for genetic diseases has been in place for at least five years2
and involves first harvesting accessible cells such as fibroblasts from the
patient and converting them into iPS cells. Next, the patient's genetic defect
is corrected in those iPS cells. Finally, the corrected iPS cells are
differentiated into the desired cell type and transplanted back into the
patient, in which the cells engraft and proliferate in the local tissue to
treat the disease.

A
key stumbling point comes at the second step, as the targeted correction of a
mutation in one gene must avoid introducing errors in surrounding genes. Many
standard methods of gene targeting leave residual errors, which makes them
unsafe for use in humans.

To
address the issue, researchers from the Wellcome Trust Sanger Institute and the University of Cambridge and colleagues
developed a gene-targeting method that combined two existing techniques-zinc
finger nucleases (ZFNs) and piggyBac transposons. The former ensures gene
targeting is efficient and accurate, and the latter that no residual genomic
errors remain.

As
proof of principle, the researchers studied α1-antitrypsin
(AAT; A1AT; SERPINA1) deficiency, an autosomal
recessive disorder that results from a mutant A1AT protein. The
deficiency leads to protein inclusion bodies in the liver and to cirrhosis.
Currently, the only therapy is a liver transplant.

The
researchers first harvested fibroblasts from A1AT-deficient patients
and converted them into iPS cells using standard methods. They then used the
ZFN and piggyBac transposon technologies to correct the mutant A1AT gene in the iPS
cells.

About
4% of the iPS cells were corrected for both mutant alleles. The genomes of the
corrected iPS cell lines proved to be relatively unaltered compared to the
genome of the parental iPS cell line. Exome sequencing of one of the corrected
cell lines detected 29 mutations in protein-coding DNA that could have occurred
during reprogramming or growth in culture.

In
their paper in Nature, the researchers
said the mutations were minor and were unlikely to disrupt any biological
functions of the cells.

Next,
the team differentiated the corrected iPS cells into hepatocyte-like cells,
which were injected into the livers of transgenic mice whose hepatocytes had
been destroyed by albumin-urokinase (Alb-uPA). The injected
cells engrafted throughout the liver and secreted human proteins without
forming tumors (see"From fibroblast to functional hepatocyte").

Philip
Gregory, SVP of research and CSO at Sangamo, and Ludovic Vallier, colead author
on the study, said the group's next steps are "to test the efficiency of
the approach to scale up for producing the corrected iPS-derived
hepatocyte-like cells and to develop a GMP protocol."

Vallier
is a principle investigator and medical research council senior fellow at the University
of Cambridge.

Pristine
wilderness

Sean Wu, assistant professor of medicine at Harvard Medical School and a principal
faculty member at the Harvard Stem Cell Institute, said using
fibroblast-derived human iPS cells to correct the A1AT genotype "was
a good first target to show proof of principle. The team's idea to use zinc
finger nuclease and piggyBac to enhance the homologous recombination in human
iPS cells is a reasonable first choice. But the zinc finger nuclease method is
not 100% site specific and may introduce additional nontargeted integrations
and mutations."

Wu
said he "would like to see if the corrected hepatocyte-like cells can
rescue an animal model of A1AT deficiency to see
if the disease can be corrected in vivo."

George
Daley, director of the stem cell transplantation program at Children's Hospital Boston and scientific
cofounder of iPS cell company iPierian Inc., thought the more important question is
safety.

Andrey
Semechkin, CEO and cochairman of International Stem Cell Corp., concurred. "Genomic
instability induced by reprogramming or genomic modification is a main issue
for the safe use of iPS cells, and all iPS cell lines will need to be analyzed
carefully before iPS technology can be successfully used in clinical
application," he told SciBX.

"It's
very important to be sure that the cells don't contain cancer-causing or other
serious types of mutations," Semechkin added. "It needs to be shown
that iPS cells-as well as any stem cell progeny-will function normally without
tumor formation for a significant period of time, which will require extensive
long-term testing in animals."

Vallier
agreed and has already extended the mouse engraftment work to 12 weeks, at
which time the animals still were tumor free.

He
also thinks that "a deeper understanding of the biological impact of
genomic changes and epigenetic instability will be crucial to achieve a high
level of safety."

"Risk
assessment might also be a factor in terms of what is considered safe. If an
individual has a terminal or debilitating disease with no therapeutic options,
well then, iPS cell therapy might become an acceptable choice," said
Daley.

The
patent and licensing status for the ZFN- and piggyBac-mediated gene correction
of human iPS cells is undisclosed.

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